PD-1 antibody secreting type PB-CAR-T cell targeting c-Met and application

Abstract

The invention discloses a PD-1 antibody secreting type PB-CAR-T cell targeting c-Met and application, and relates to the technical field of tumor immunotherapy, a chimeric antigen receptor CAR sequence containing a PD-1 single-chain antibody scFv and c-Met scFv is integrated into a T cell genome through a PiggyBac transposon system, and the CAR sequence comprises a PD-1 antibody secreting structural domain and a c-Met antigen binding structural domain; a PD-1 antibody secretion structural domain and a c-Met targeted CAR sequence are efficiently integrated into a T cell through a PiggyBac transposon system non-viral vector, the multiplication capacity, tumor killing efficiency and memory phenotype proportion of the CAR-T cell are remarkably improved, meanwhile, immune checkpoints are blocked through local secretion of a PD-1 antibody, immunosuppression in a solid tumor microenvironment is overcome, the risk of immune escape is reduced, and the CAR-T cell has a good application prospect. Compared with a traditional lentiviral vector, the method has the advantages of being high in safety, low in cost and simplified in preparation process, and a new strategy with more clinical transformation potential is provided for treatment of the c-Met positive solid tumor.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to a PD-1 antibody-secreting PB-CAR-T cell targeting c-Met and its application. Background Technology

[0002] The PiggyBac transposon system, as a non-viral vector, has advantages such as non-infectiousness, low cost, stable integration, and high load capacity, and has been proven to efficiently deliver CAR sequences to T cells. Simultaneously, secretory PD-1 antibodies can locally block the PD-1 / PD-L1 pathway, enhancing CAR-T cell activity.

[0003] In the field of tumor immunotherapy, traditional lentiviral vectors have limitations in vector capacity when preparing bispecific CAR-T (such as c-Met / PD-1CAR-T), making it difficult to stably express long CAR sequences. Viral vectors also have problems such as potential infection risks, high production costs, and complex processes, which limit the popularization of CAR-T therapy. In solid tumors, activation of the PD-1 / PD-L1 pathway leads to CAR-T cell depletion, requiring the combination of immune checkpoint blockade strategies. However, traditional combination methods have problems such as spatial structure mismatch or low gene editing efficiency. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a PD-1 antibody-secreting PB-CAR-T cell targeting c-Met to address the limitations of traditional lentiviral vectors in preparing bispecific CAR-T cells (such as c-Met / PD-1 CAR-T), including vector capacity limitations, difficulty in stably expressing long CAR sequences, potential infection risks, high production costs, and complex processes. These issues restrict the widespread use of CAR-T therapy. In solid tumors, PD-1 / PD-L1 pathway activation leads to CAR-T cell depletion, requiring combined immune checkpoint blockade strategies. However, traditional combined methods suffer from spatial mismatch or low gene editing efficiency.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a PD-1 antibody-secreting PB-CAR-T cell targeting c-Met, comprising: A chimeric antigen receptor (CAR) sequence containing a PD-1 single-chain antibody scFv and a c-Met scFv was integrated into the T cell genome using the PiggyBac transposon system. The CAR sequence contains a PD-1 antibody secretion domain and a c-Met antigen-binding domain.

[0007] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, wherein the PiggyBac transposon system comprises the recombinant plasmid PB-CP CAR and the transposase plasmid PB200PA-1, which are co-transfected into activated T cells via electroporation.

[0008] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, wherein: the CAR-T cells have CD8+ on their surface + T cell proportion ≥84%, central memory T cell CCR7 + CD45RO + The proportion is ≥47.5%, and it can secrete PD-1 antibodies to block the PD-1 / PD-L1 pathway.

[0009] Secondly, the present invention provides a method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met, comprising: S1. Insert the CAR sequences of PD-1 scFv and c-Met scFv into the PiggyBac transposon vector PB513B-1 to obtain the recombinant plasmid PB-CP CAR; S2. Mix PB-CP CAR with transposase plasmid PB200PA-1 at a 1:1 ratio and co-electroporate with activated T cells; S3. Positive cells were screened by flow cytometry and PB-CP CAR-T cells were obtained by in vitro expansion.

[0010] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, the electroporation conditions are as follows: using a Lonza 4D-Nucleofector™ electroporator, the program is E0138-B1-mix, and the transfection efficiency is ≥36.1%.

[0011] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, in step S1, the CAR sequences of PD-1 scFv and c-Met scFv are inserted into the PB513B-1 vector by Infusion cloning and verified by enzyme digestion.

[0012] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, in step S2, the activated T cells are PBMCs isolated from the peripheral blood of healthy volunteers by the Ficoll density gradient method and activated with CD3 / CD28 antibody for 48 hours.

[0013] As a preferred embodiment of the PD-1 antibody-secreting PB-CAR-T cells targeting c-Met described in this invention, in step S3, the screening of positive cells is achieved by detecting EGFP expression by flow cytometry, and the amplification culture medium contains IL-2.

[0014] Thirdly, the present invention provides an application of PD-1 antibody-secreting PB-CAR-T cells targeting c-Met, wherein the PB-CAR-T cells are used to prepare drugs for treating c-Met-positive solid tumors, including lung adenocarcinoma.

[0015] Fourthly, the present invention provides an application of PD-1 antibody-secreting PB-CAR-T cells targeting c-Met, characterized in that: the PB-CAR-T cells enhance the killing ability of T cells against tumor cells and reduce immune escape by secreting PD-1 antibodies and targeting c-Met antigens.

[0016] The beneficial effects of this invention are as follows: By efficiently integrating the PD-1 antibody secretory domain and the c-Met-targeting CAR sequence into T cells through the PiggyBac transposon system non-viral vector, the proliferation capacity, tumor killing efficiency, and memory phenotype ratio of CAR-T cells are significantly enhanced. At the same time, by locally secreting PD-1 antibodies to block immune checkpoints, the immunosuppression in the solid tumor microenvironment is overcome, and the risk of immune escape is reduced. Compared with traditional lentiviral vectors, this method has the advantages of high safety, lower cost, and simplified preparation process, providing a new strategy with greater clinical translational potential for the treatment of c-Met-positive solid tumors. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart of PD-1 antibody-secreting PB-CAR-T cells targeting c-Met in Example 1.

[0019] Figure 2 This is a schematic diagram of the recombinant vector PB-CP CAR spectrum and identification in Example 1.

[0020] Figure 3 This is a schematic diagram illustrating the flow cytometry detection of c-Met / PD-1 CAR-T cell infection efficiency in Example 1; In the diagram: A, activated T cells; B, Lenti CAR-T; C, PB-CP CAR-T.

[0021] Figure 4 This is a schematic diagram comparing the proliferation activities of PB-CP CAR-T, Lenti CAR-T, activated T cells, and cancer cells after co-culturing in Example 1.

[0022] Figure 5 This is a schematic diagram comparing the killing efficiency of PB-CP CAR-T, Lenti CAR-T, activated T cells and cancer cells after co-culturing in Example 1.

[0023] Figure 6 This is a schematic diagram showing the proportion of cell subtypes in each group of CAR-T cells detected by flow cytometry in Example 1; In the image: A, ActiveT cells; B, Lenti CAR-T; C, PB-CP CAR-T.

[0024] Figure 7 This is a schematic diagram illustrating the changes in the phenotype of memory cells in each group of CAR-T cells before and after activation by cancer cells, as detected by flow cytometry in Example 1.

[0025] Figure 8 This is a schematic diagram illustrating the apoptosis status of CAR-T cells in each group during flow cytometry analysis in Example 1. In the image: A, ActiveT cells; B, Lenti CAR-T; C, PB-CP CAR-T.

[0026] Figure 9 This is a schematic diagram illustrating the activation status of CAR-T cells stimulated by cancer cells in each group using flow cytometry in Example 1. In the image: A, ActiveT cells; B, Lenti CAR-T; C, PB-CP CAR-T.

[0027] Figure 10 This is a schematic diagram illustrating the gene expression of CAR-T cells stimulated by cancer cells in each group, as detected by transcriptome sequencing in Example 1. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] Example 1, referring to Figure 1 - Figure 10 This is the first embodiment of the present invention, which provides PD-1 antibody-secreting PB-CAR-T cells targeting c-Met and their preparation method, including: A chimeric antigen receptor (CAR) sequence containing a PD-1 single-chain antibody scFv and a c-Met scFv was integrated into the T cell genome using the PiggyBac transposon system. The CAR sequence contains a PD-1 antibody secretion domain and a c-Met antigen-binding domain. The PiggyBac transposon system contains the recombinant plasmid PB-CP CAR and the transposase plasmid PB200PA-1, which are co-transfected into activated T cells via electroporation. CAR-T cell surface CD8 + T cell proportion ≥84%, central memory T cell CCR7 + CD45RO + The proportion is ≥47.5%, and it can secrete PD-1 antibodies to block the PD-1 / PD-L1 pathway; Preparation methods include: S1. Construction of recombinant plasmid: Insert the CAR sequences of PD-1 scFv and c-Met scFv into the PiggyBac transposon vector PB513B-1 to obtain the recombinant plasmid PB-CP CAR; Furthermore, in step S1, the CAR sequences of PD-1 scFv and c-Met scFv are inserted into the PB513B-1 vector using the Infusion cloning method and verified by enzyme digestion. It should be noted that in step S1, the Infusion cloning method ensures the precise ligation of the CAR sequences of PD-1 scFv and c-Met scFv with the PB513B-1 vector. This method offers advantages such as high efficiency and seamless splicing, avoiding the extra bases that may be introduced by traditional enzyme digestion ligation. Enzyme digestion verification was performed using EcoRI and BamHI double digestion and electrophoresis analysis, confirming the successful construction of the recombinant plasmid PB-CP CAR and confirming that no vector self-ligation or insertion fragment loss occurred.

[0032] S2. Electroporation transfection: PB-CP CAR and transposase plasmid PB200PA-1 were mixed at a 1:1 ratio and co-electroplated with activated T cells. Furthermore, the electroporation conditions were as follows: using a Lonza 4D-Nucleofector™ electroporator, with program E0138-B1-mix, and transfection efficiency ≥36.1%; In step S2, the activated T cells are PBMCs isolated from the peripheral blood of healthy volunteers using the Ficoll density gradient method and activated with CD3 / CD28 antibody for 48 hours. It should be noted that in step S2, the Lonza 4D-Nucleofector™ electroporator program E0138-B1-mix has been optimized to significantly improve transfection efficiency (≥36.1%) while reducing cell damage. Activated T cells need to be stimulated with CD3 / CD28 antibodies for 48 hours to achieve optimal transfection status, at which time the cells are in the proliferation phase and their membrane permeability is enhanced, which is conducive to efficient plasmid delivery.

[0033] S3. Screening and Expansion: Positive cells were screened by flow cytometry and expanded in vitro to obtain PB-CP CAR-T cells; Furthermore, in step S3, positive cells are screened by detecting EGFP expression using flow cytometry, and the amplification medium contains IL-2. It should be noted that in step S3, EGFP, as a reporter gene, can be used to rapidly screen positive cells via flow cytometry, ensuring the homogeneity of the subsequently expanded CAR-T cell population. The addition of IL-2 (100 IU / mL) to the culture medium can maintain T cell activity and proliferation capacity. The required number of cells for treatment (≥1×10⁻⁶) can be obtained 7 days after expansion. 8 (number), and the cell phenotype is stable (CD8). + ≥84%, central memory T cells ≥47.5%.

[0034] In summary, this invention efficiently integrates the PD-1 antibody secretory domain and the c-Met-targeting CAR sequence into T cells via a non-viral vector using the PiggyBac transposon system. This significantly enhances the proliferation capacity, tumor-killing efficiency, and memory phenotype ratio of CAR-T cells. Simultaneously, by locally secreting PD-1 antibodies to block immune checkpoints, it overcomes immunosuppression in the solid tumor microenvironment and reduces the risk of immune escape. Compared to traditional lentiviral vectors, this method offers advantages such as high safety, lower cost, and simplified preparation process, providing a new strategy with greater clinical translational potential for the treatment of c-Met-positive solid tumors.

[0035] Example 2, refer to Figure 2 - Figure 10This is the second embodiment of the present invention, which provides a method for preparing and functionally validating PD-1 antibody-secreting CAR-T cells targeting c-Met based on the PiggyBac transposon (PB-CP CAR-T), specifically including: Construction of recombinant plasmids ( Figure 2 Linearized fragments of the PB513B-1 vector were obtained by double digestion with EcoRI and BamHI. The CAR sequences of PD-1 scFv and c-Met scFv were inserted into the vector using the In-Fusion cloning method. After transformation into STBL3 competent cells, plasmids were extracted, and the recombinant plasmid PB-CP CAR was obtained by sequencing and restriction enzyme digestion verification. Restriction digestion electrophoresis results showed that lane 1 contained the PB-CP CAR double digestion product with EcoRI and BamHI, lane 2 contained the intact recombinant plasmid, and lane 3 contained the DNA marker (…). Figure 2 B).

[0036] T cell transfection ( Figure 3 ): PBMCs were isolated from peripheral blood of healthy volunteers, activated with CD3 / CD28 antibody for 48 hours, and then 1 μg of PB-CP CAR was mixed with 1 μg of PB200PA-1 plasmid. The mixture was then transfected 2 × 10⁶ cells using a Lonza 4D-Nucleofector™ electroporator (program E0138-B1-mix). 6 Flow cytometry analysis showed a transfection efficiency of 36.1% for 1,000 T cells. Figure 3 C), significantly higher than the lentiviral vector group (28.3%).

[0037] Cell expansion: After transfection, cells were cultured in IL-2 (100 IU / mL) medium for 7 days, and EGFP was sorted by flow cytometry. + Cell expansion, obtaining CD8 in PB-CP CAR-T cells + The proportion of T cells reached 84%, with central memory T cells (CCR7) + CD45RO + ) proportion 47.5% Figure 6-7 ).

[0038] The details are shown in Table 1 below:

[0039] Table 1: Summary Table of Key Experimental Data Functionality verification: Proliferative capacity ( Figure 4 ): CCK-8 assay showed that after co-culturing with A549 cells for 5 days, the proliferation of PB-CP CAR-T cells was 1.5 times that of the lentivirus group (P<0.001). Kill efficiency ( Figure 5At an efficacy-to-target ratio of 20:1, the killing rates against A549 and H1975 cells were 43.49±4.37% and 42.44±2.50%, respectively, which were approximately 10% higher than those against the lentivirus group. Safety: Kill rate of c-Met-negative HBSMC cells <5% ( Figure 4-5 ); Gene expression ( Figure 10 Transcriptome analysis showed that CX3CR1 and GNLY genes were significantly upregulated, and the immune infiltration score was increased by 30%.

[0040] Application effect: PB-CP CAR-T cells were used at a rate of 1×10⁻⁶. 6 Intravenous injection of a dose of / kg into lung adenocarcinoma model mice reduced tumor volume by 62.3% compared to the control group (P<0.001), and no side effects such as cytokine storm were observed.

[0041] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A PD-1 antibody-secreting PB-CAR-T cell targeting c-Met, characterized in that: include: A chimeric antigen receptor (CAR) sequence containing a PD-1 single-chain antibody scFv and a c-Met scFv was integrated into the T cell genome using the PiggyBac transposon system. The CAR sequence contains a PD-1 antibody secretion domain and a c-Met antigen-binding domain.

2. The PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 1, characterized in that: The PiggyBac transposon system comprises the recombinant plasmid PB-CP CAR and the transposase plasmid PB200PA-1, which are co-transfected into activated T cells via electroporation.

3. The PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 2, characterized in that: CD8 on the surface of CAR-T cells + T cell proportion ≥84%, central memory T cell CCR7 + CD45RO + The proportion is ≥47.5%, and it can secrete PD-1 antibodies to block the PD-1 / PD-L1 pathway.

4. A method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met, as described in claims 1-3, characterized in that: include: S1. Insert the CAR sequences of PD-1 scFv and c-Met scFv into the PiggyBac transposon vector PB513B-1 to obtain the recombinant plasmid PB-CP CAR; S2. Mix PB-CP CAR with transposase plasmid PB200PA-1 at a 1:1 ratio and co-electroporate with activated T cells; S3. Positive cells were screened by flow cytometry and PB-CP CAR-T cells were obtained by in vitro expansion.

5. The method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 4, characterized in that: The electroporation conditions were as follows: using a Lonza 4D-Nucleofector™ electroporator, program E0138-B1-mix, and transfection efficiency ≥36.1%.

6. The method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 5, characterized in that: In step S1, the CAR sequences of PD-1 scFv and c-Met scFv are inserted into the PB513B-1 vector using the Infusion cloning method and verified by enzyme digestion.

7. The method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 6, characterized in that: In step S2, the activated T cells are PBMCs isolated from the peripheral blood of healthy volunteers using the Ficoll density gradient method and activated with CD3 / CD28 antibody for 48 hours.

8. The method for preparing PD-1 antibody-secreting PB-CAR-T cells targeting c-Met as described in claim 7, characterized in that: In step S3, positive cells are screened by detecting EGFP expression using flow cytometry, and the amplification medium contains IL-2.

9. The application of a PD-1 antibody-secreting PB-CAR-T cell targeting c-Met, characterized in that: The PB-CAR-T cells are used to prepare drugs for treating c-Met-positive solid tumors, including lung adenocarcinoma.

10. The application of a PD-1 antibody-secreting PB-CAR-T cell targeting c-Met, characterized in that: The PB-CAR-T cells enhance the killing ability of T cells against tumor cells and reduce immune escape by secreting PD-1 antibodies and targeting c-Met antigens.

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